Plants having improved growth characteristics and method for making the same

ABSTRACT

The present invention concerns a method for improving growth characteristics of plants by increasing expression and/or activity in a plant of an LRR receptor kinase or a homologue thereof. One such method comprises introducing into a plant an RLK827 nucleic acid molecule or functional variant thereof. The invention also relates to transgenic plants having improved growth characteristics, which plants have modulated expression of a nucleic acid encoding an LRR receptor kinase. The present invention also concerns constructs useful in the methods of the invention.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/632,570 which is national stage application (under 35 U.S.C. 371)of PCT/EP2005/053397 filed Jul. 14, 2005, which claims benefit ofEuropean application 04103393.7 filed Jul. 15, 2004 and U.S. Provisionalapplication 60/589,235 filed Jul. 20, 2004. The entire content of eachabove-mentioned application is hereby incorporated by reference in itsentirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_Listing_(—)14546_(—)00075_US. The sizeof the text file is 146 KB, and the text file was created on Jun. 27,2011.

BACKGROUND OF INVENTION

The present invention relates generally to the field of molecularbiology and concerns a method for improving plant growthcharacteristics. More specifically, the present invention concerns amethod for increasing yield and/or biomass of a plant by increasing theexpression and/or activity of an LRR receptor kinase (RLK827) or ahomologue thereof in a plant. The present invention also concerns plantshaving increased expression of a nucleic acid encoding an LRR receptorkinase or a homologue thereof, which plants have improved growthcharacteristics relative to corresponding wild type plants. Theinvention also provides constructs useful in the methods of theinvention.

Given the ever-increasing world population, and the dwindling area ofland available for agriculture, it remains a major goal of agriculturalresearch to improve the efficiency of agriculture and to increase thediversity of plants in horticulture. Conventional means for crop andhorticultural improvements utilise selective breeding techniques toidentify plants having desirable characteristics. However, suchselective breeding techniques have several drawbacks, namely that thesetechniques are typically labour intensive and result in plants thatoften contain heterogeneous genetic complements that may not alwaysresult in the desirable trait being passed on from parent plants.Advances in molecular biology have allowed mankind to manipulate thegermplasm of animals and plants. Genetic engineering of plants entailsthe isolation and manipulation of genetic material (typically in theform of DNA or RNA) and the subsequent introduction of that geneticmaterial into a plant. Such technology has led to the development ofplants having various improved economic, agronomic or horticulturaltraits. Traits of particular economic interest are growthcharacteristics such as high yield. Yield is normally defined as themeasurable produce of economic value from a crop. This may be defined interms of quantity and/or quality. Yield is directly, dependent onseveral factors, for example, the number and size of the organs, plantarchitecture (for example, the number of branches), seed production andmore. Root development, nutrient uptake and stress tolerance may also beimportant factors in determining yield. Crop yield may therefore beincreased by optimising one of the abovementioned factors.

Growth and development of plants is determined by environmental andinternal signals, such as hormone mediated signalling, stress andnutrient signalling, cell cycle control or developmental signalling.Cells perceive these signals via cell surface receptors, which transducethe signal to the inside of the cell. Many of these receptors areprotein kinases. Protein kinases comprise a large family of enzymes thatmediate the response of eukaryotic cells to stimuli by phosphorylationof hydroxyamino acids. The enzymes fall into two broad classes withrespect to their substrate specificity: serine/threonine specific ortyrosine specific enzymes. Kinases involved in signal transduction maybe classified into different families which are mostly made up oftyrosine kinases. Receptor Tyrosine Kinases (RTK) in animals have auniform structure and are composed of an extracellular ligand bindingdomain, a transmembrane domain and a cytoplasmic tyrosine kinase domain.Among the plant tyrosine kinases, the Receptor-Like Kinase (RLK)proteins take a prominent place. More than 600 different RLKs are knownin plants. They have a similar structure as the animal RTKs, aclassification is given in FIG. 1 (Shiu and Bleecker, Proc. Natl. Acad.Sci. USA 98, 10763-10768, 2001). Several plant RLK proteins have beencharacterised, for example BRH (brassinoid signalling), CLV1 (meristemdifferentiation), HAESA (abscission of floral organs), XA21 (fungaldetection) CR4 (leaf and endosperm development), FLS2(flagellin/pathogen detection), SRK (self-incompatibility), among others(Becraft, Annu. Rev. Cell Dev. Biol. 18, 163-192, 2002; Dievart andClark, Curr. Opin. Plant Biol. 6, 507-516). About 200 of the plant RLKspossess a Leucine Rich Repeat (LRR). LRRs are sequence motifs of 23 to25 residues, which comprise a consensus sequence LxxLxLxxN/CxL wherein xmay be any amino acid. These LRRs are present in proteins with diversefunctions, such as hormone receptor interactions, enzyme inhibition,cell adhesion and cellular trafficking and frequently the LRR domainsare organised in tandem arrays. It was shown that LRRs may be criticalfor the morphology and dynamics of the cytoskeleton. The primaryfunction of these motifs appears to be providing a versatile structuralframework for the formation of protein-protein interactions (Kobe andKajava, Curr. Opin. Struct. Biol. 11, 725-732, 2001).

The combination of Leucine Rich Repeats and kinase domains ischaracteristic for receptor proteins that mediate external signals intothe cell. They are thought to act by a mechanism in which the LRRdomain(s), mostly extracellular, act as a sensor for an extracellularsignal whereas the kinase domain is usually internal and participates inthe transduction of the signal by phosphorylating intracellular targetsand thus initiating the signal transduction. RLKs have been implicatedin plants in a variety of process like plant development, diseaseresistance or self-incompatibility. It is shown in this invention thatplant growth characteristics, and in particular yield, may be improvedby modulating expression in a plant of a nucleic acid encoding an RLK.

International patent application WO 03/072763 disclosed a receptor likekinase which, when overexpressed in plants, resulted in increased plantgrowth and seed production. However, the subject RLK protein did notcomprise any LRR domains in its non-cytoplasmic domain, but instead thisdomain was Proline rich. Another disclosure (WO 00/04761) reported thatupon overexpression of the RKN receptor kinase, root growth wasenhanced. Similarly, it was suggested, but not shown, in WO 98/59039that overexpression of the BRH receptor kinase would result in modulatedyield. However the RLK used in the latter two cases comprised 22 LRRdomains in the non-cytoplasmic domain, typical for the LRR-X subfamilyof receptor like kinases. So far there have been no reports to show oreven suggest that receptor like kinases of the LRR-I subfamily may beuseful for improving plant growth characteristics, and in particular inincreasing yield.

It has now surprisingly been found that increasing expression and/oractivity, relative to corresponding wild type plants, of an RLK827protein in plants gives plants having improved growth characteristics,and in particular increased yield.

RLK827 is a receptor like kinase that is structurally related to LRRPK1which is a member of the LRR-I subfamily of receptor like kinases (Shiuand Bleecker, 2001). The mature RLK827 protein has, starting from theN-terminus, a long putative non-cytoplasmic domain, a singletransmembrane domain and a kinase domain in the C-terminal cytoplasmicpart. The receptor like kinases are classified according to thecomposition of their non-cytoplasmic domain, which may comprise prolinerich sequences, lectin domains, LRR domains, EGF repeats, TNFR repeats,thaumatin or agglutinin domains etc. A large group of receptor likekinases have Leucine Rich Repeats (LRR) in the non-cytoplasmic domain.The various LRR subfamilies differ from each other0 in the number andposition of these leucine rich repeats (for an overview, see Shiu andBleecker, 2001). The putative non-cytoplasmic domain of RLK827 ischaracterised by the presence of one up to three tandem leucine richrepeat domains in its C-terminal part; RLK827 therefore belongs in thesubfamily of LRR-I receptor kinases. The various LRR subfamilies ofreceptor kinases also differ from one other in their chromosomaldistribution. Often they are arranged in tandem repeats. Tandemduplications, large-scale duplications and rearrangements of chromosomesare, at least in part, responsible for the evolution and expansion ofthe LRR receptor like kinases in plants. Accordingly, with a fewexceptions, LRR-I receptor kinases are distributed on chromosome I, IIand III in Arabidopsis.

According to one embodiment of the present invention there is provided amethod for improving growth characteristics of a plant comprisingincreasing expression and/or activity of an RLK827 polypeptide or ahomologue thereof and optionally selecting for plants having improvedgrowth characteristics.

Advantageously, performance of the methods according to the presentinvention result in plants having a variety of improved growthcharacteristics, such as improved growth, improved yield, improvedbiomass, modified architecture or improved cell division, each relativeto corresponding wild type plants. Preferably, the improved growthcharacteristics comprise at least increased yield relative tocorresponding wild type plants. Preferably, the increased yield isincreased seed yield, which includes increased number of (filled) seeds,increased total weight of seeds and increased harvest index.

The term “increased yield” as defined herein is taken to mean anincrease in any one or more of the following, each relative tocorresponding wild type plants: (i) increased biomass (weight) of one ormore parts of a plant, particularly aboveground (harvestable) parts,increased root biomass or increased biomass of any other harvestablepart; (ii) increased total seed yield, which includes an increase inseed biomass (seed weight) and which may be an increase in the seedweight per plant (total seed weight) or on an individual seed basis;(iii) increased number of (filled) seeds; (iv) increased seed size; (v)increased seed volume; (vi) increased individual seed area; (vii)increased individual seed length; (viii) increased harvest index, whichis expressed as a ratio of the yield of harvestable parts, such asseeds, over the total biomass; (ix) increased number of florets perpanicle which is extrapolated from the total number of seeds counted andthe number of primary panicles; and (x) increased thousand kernel weight(TKW), which is extrapolated from the number of filled seeds counted andtheir total weight. An increased TKW may result from an increased seedsize (length, width or both) and/or seed weight. An increased TKW mayresult from an increase in embryo size and/or endosperm size.

Taking corn as an example, a yield increase may be manifested as one ormore of the following: increase in the number of plants per hectare oracre, an increase in the number of ears per plant, an increase in thenumber of rows, number of kernels per row, kernel weight, TKW, earlength/diameter, among others. Taking rice as an example, a yieldincrease may be manifested by an increase in one or more of thefollowing: number of plants per hectare or acre, number of panicles perplant, number of spikelets per panicle, number of flowers per panicle,increase in the seed filling rate, increase in TKW, among others. Anincrease in yield may also result in modified architecture, or may occuras a result of modified architecture.

Preferably, performance of the methods of the present invention resultsin plants having increased yield and/or increased biomass. Moreparticularly, performance of the methods according to the presentinvention results in plants having increased seed yield. Preferably, theincreased seed yield comprises an increase in one or more of number offilled seeds, total seed weight, and harvest index, each relative tocontrol plants. Therefore, according to the present invention, there isprovided a method for increasing plant yield, which method comprisesincreasing expression and/or activity in a plant of an RLK827polypeptide or a homologue thereof.

Since the modified plants according to the present invention haveincreased yield, it is likely that these plants exhibit an increasedgrowth rate (during at least part of their life cycle), relative to thegrowth rate of corresponding wild type plants at a corresponding stagein their life cycle. The increased growth rate may be specific to one ormore parts or cell types of a plant (including seeds), or may bethroughout substantially the whole plant. Plants having an increasedgrowth rate may have a shorter life cycle. The life cycle of a plant maybe taken to mean the time needed to grow from a dry mature seed up tothe stage where the plant has produced dry mature seeds, similar to thestarting material. This life cycle may be influenced by factors such asearly vigour, growth rate, flowering time and speed of seed maturation.An increase in growth rate may take place at one or more stages in thelife cycle of a plant or during substantially the whole plant lifecycle. Increased growth rate during the early stages in the life cycleof a plant may reflect enhanced vigour. The increase in growth rate mayalter the harvest cycle of a plant allowing plants to be sown laterand/or harvested sooner than would otherwise be possible. If the growthrate is sufficiently increased, it may allow for the sowing of furtherseeds of the same plant species (for example sowing and harvesting ofrice plants followed by sowing and harvesting of further rice plants allwithin one conventional growing period). Similarly, if the growth rateis sufficiently increased, it may allow for the sowing of further seedsof different plants species (for example the sowing and harvesting ofrice plants followed by, for example, the sowing and optional harvestingof soy bean, potatoes or any other suitable plant). Harvestingadditional times from the same rootstock in the case of some plants mayalso be possible. Altering the harvest cycle of a plant may lead to anincrease in annual biomass production per acre (due to an increase inthe number of times (say in a year) that any particular plant may begrown and harvested). An increase in growth rate may also allow for thecultivation of transgenic plants in a wider geographical area than theirwild-type counterparts, since the territorial limitations for growing acrop are often determined by adverse environmental conditions either atthe time of planting (early season) or at the time of harvesting (lateseason). Such adverse conditions may be avoided if the harvest cycle isshortened. The growth rate may be determined by deriving variousparameters from growth curves plotting growth experiments, suchparameters may be: T-Mid (the time taken for plants to reach 50% oftheir maximal size) and T-90 (time taken for plants to reach 90% oftheir maximal size), amongst others.

Performance of the methods of the invention gives plants having anincreased growth rate. Therefore, according to the present invention,there is provided a method for increasing the growth rate of plants,which method comprises increasing expression and/or activity in a plantof an RLK827 polypeptide or a homologue thereof.

An increase in yield and/or growth rate occurs whether the plant isunder non-stress conditions or whether the plant is exposed to variousstresses compared to control plants. Plants typically respond toexposure to stress by growing more slowly. In conditions of severestress, the plant may even stop growing altogether. Mild stress on theother hand is defined herein as being any stress to which a plant isexposed which does not result in the plant ceasing to grow altogetherwithout the capacity to resume growth. Due to advances in agriculturalpractices (irrigation, fertilization, pesticide treatments) severestresses are not often encountered in cultivated crop plants. As aconsequence, the compromised growth induced by mild stress is often anundesirable feature for agriculture. Mild stresses are the typicalstresses to which a plant may be exposed. These stresses may be theeveryday biotic and/or abiotic (environmental) stresses to which a plantis exposed. Typical abiotic or environmental stresses includetemperature stresses caused by atypical hot or cold/freezingtemperatures; salt stress; water stress (drought or excess water).Abiotic stresses may also be caused by chemicals. Biotic stresses aretypically those stresses caused by pathogens, such as bacteria, viruses,fungi and insects.

The abovementioned growth characteristics may advantageously be modifiedin any plant.

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,leaves, roots (including tubers), flowers, and tissues and organs,wherein each of the aforementioned comprise the gene/nucleic acid ofinterest or the specific modification in the gene/nucleic acid ofinterest. The term “plant” also encompasses plant cells, suspensioncultures, callus tissue, embryos, meristematic regions, gametophytes,sporophytes, pollen, and microspores, again wherein each of theaforementioned comprise the gene/nucleic acid of interest.

Plants that are particularly useful in the methods of the inventioninclude algae, ferns, and all plants which belong to the superfamilyViridiplantae, in particular monocotyledonous and dicotyledonous plants,including fodder or forage legumes, ornamental plants, food crops,trees, or shrubs selected from the list comprising Abelmoschus spp.,Acer spp., Actinidia spp., Agropyron spp., Allium spp., Amaranthus spp.,Ananas comosus, Annona spp., Apium graveolens, Arabidopsis thaliana,Arachis spp, Artocarpus spp., Asparagus officinalis, Avena sativa,Averrhoa carambola, Benincasa hispida, Bertholletia excelsea, Betavulgaris, Brassica spp., Cadaba farinosa, Camellia sinensis, Cannaindica, Capsicum spp., Carica papaya, Carissa macrocarpa, Carthamustinctorius, Carya spp., Castanea spp., Cichorium endivia, Cinnamomumspp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colaspp., Colocasia esculenta, Corylus spp., Crataegus spp., Cucumis spp.,Cucurbita spp., Cynara spp., Daucus carota, Desmodium spp., DimocarpusIongan, Dioscorea spp., Diospyros spp., Echinochloa spp., Eleusinecoracana, Eriobotrya japonica, Eugenia uniflora, Fagopyrom spp., Fagusspp., Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba,Glycine spp., Gossypium hirsutum, Helianthus spp., Hibiscus spp.,Hordeum spp., Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrusspp., Lemna spp., Lens culinaris, Linum usitatissimum, Litchi chinensis,Lotus spp., Luffa acutangula, Lupinus spp., Macrotyloma spp., Malpighiaemarginata, Malus spp., Mammea americana, Mangifers indica, Manihotspp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp.,Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp.,Opuntia spp., Ornithopus spp., Oryza spp., Panicum miliaceum, Passifloraedulis, Pastinaca sativa, Persea spp., Petroselinwn crispum, Phaseolusspp., Phoenix spp., Physalis spp., Pinus spp., Pistacia vera, Pisumspp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp.,Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheumrhabarbarum, Ribes spp., Rubus spp., Saccharum spp., Sambucus spp.,Secale cereale, Sesamum spp., Solanum spp., Sorghum bicolor, Spinaciaspp., Syzygium spp., Tamarindus indica, Theobroma cacao, Trifolium spp.,Triticosecale rimpaui, Triticum spp., Vaccinium spp., Vicia spp., Vignaspp., Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amongstothers.

According to a preferred feature of the present invention, the plant isa crop plant comprising soybean, sunflower, canola, alfalfa, rapeseed orcotton. Further preferably, the plant according to the present inventionis a monocotyledonous plant such as sugarcane, most preferably a cereal,such as rice, maize, wheat, millet, barley, oats or sorghum.

The activity of an RLK827 protein may be increased by increasing levelsof the RLK827 polypeptide. Alternatively, activity may also be increasedwhen there is no change in levels of an RLK827, or even when there is areduction in levels of an RLK827. This may occur when the intrinsicproperties of the polypeptide are altered, for example, by making amutant or selecting a variant that is more active that the wild type.

The term “RLK827 or homologue thereof” as defined herein refers to aReceptor Like Kinase (RLK) having kinase activity and comprising in itsmature form a non-cytoplasmic domain (extracellular domain), a singletransmembrane domain and a putative cytoplasmic kinase domain. Thenon-cytoplasmic domain or RLK827 comprises at least 1 but no more than 3Leucine Rich Repeat (LRR) domains, preferably two LRR domains arepresent, more preferably three LRR domains. Further preferably, thelength of the non-cytoplasmic domain ranges between 250 and 550 aminoacids. The non-cytoplasmic domain preferably comprises the amino acidsequence motif LRxFP(E/D)GxRNC(Y/F) (SEQ ID NO: 33), wherein x may beany amino acid and where up to 2 other amino acids may be replaced by aconserved substitution as listed in Table 2. Preferably, the first x inthis motif is Y or A, and the second x preferably is one of V, F, E andA.

The term “RLK827 or homologue thereof also refers to amino acidsequences having in increasing order of preference at least 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%,97%, 98% or 99% overall sequence identity to the amino acid representedby SEQ ID NO: 2.

The term “RLK827 or homologue thereof comprises RLK827 (SEQ ID NO 2),its paralogues and orthologues. The overall sequence identity isdetermined using a global alignment algorithm, such as the NeedlemanWunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys),using default parameter settings.

The various structural domains in an RLK827 protein may be identifiedusing specialised databases e.g. SMART (Schultz et al. (1998) Proc.Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic AcidsRes 30, 242-244; smart.embl-heidelberg.de webpage) or Pfam (Bateman etal., Nucleic Acids Research 30(1):276-280 (2002),sanger.ac.uk/Software/Pfam/webpage).

The kinase domain is of a STYKc type (SMART accession number SM00221,Interpro accession number IPRO04040) and has possibly dual-specificitySer/Thr/Tyr kinase activity. In the N-terminal extremity of thecatalytic domain there is a glycine-rich stretch of residues in thevicinity of a lysine residue, which has been shown to be involved in ATPbinding. In the central part of the catalytic domain there is aconserved aspartic acid residue, which is important for the catalyticactivity of the enzyme.

Furthermore, LRR domains are well known in the art and are defined inPfam (accession PF00560) as 20 to 29-residue sequence motifs present intandem arrays in a number of proteins with diverse functions, such ashormone receptor interactions, enzyme inhibition, cell adhesion andcellular trafficking. Recent studies revealed the involvement of LRRproteins in early mammalian development, neural development, cellpolarization, regulation of gene expression and apoptosis signalling.The primary function of these motifs appears to be to provide aversatile structural framework for the formation of protein-proteininteractions. Sequence analyses of LRR proteins suggested the existenceof several different subfamilies of LRRs. Apparently the repeats fromdifferent subfamilies never occur simultaneously and most probablyevolved independently. However, all major classes of LRR seem to havecurved horseshoe structures with a parallel beta sheet on the concaveside and mostly helical elements on the convex side. At least sixfamilies of LRR proteins, characterised by different lengths andconsensus sequences of the repeats, have been identified. Eleven-residuesegments of the LRRs (LxxLxLxxN/CxL), corresponding to the β-strandandadjacent loop regions, are usually conserved in LRR proteins, whereasthe remaining parts of the repeats may be very different. Despite thedifferences, each of these variable parts contains two half-turns atboth ends and a “linear” segment (as the chain follows a linear pathoverall), usually formed by a helix, in the middle. The concave face andthe adjacent loops are the most common protein interaction surfaces onLRR proteins. 3D structures of some LRR protein-ligand complexes showthat the concave surface of LRR domain is ideal for interaction withalpha-helices, thus supporting earlier conclusions that the elongatedand curved LRR structure provides an outstanding framework for achievingdiverse protein-protein interactions. Molecular modelling suggests thatthe pattern LxxLxL, which is often conserved and which is shorter thanthe previously proposed LxxLxLxxN/CxL, is sufficient to impart thecharacteristic horseshoe curvature to proteins with 20- to 30-residuerepeats. LRR domains of an LRK827 protein may differ from the canonicalLRR domains known in the art but may be identified by suitable computeralgorithms, preferably those used in the Pfam database.

Transmembrane domains are about 15 to 30 amino acids long and areusually composed of hydrophobic residues that form an alpha helix. Theyare usually predicted on the basis of hydrophobicity (for example Kleinet al., Biochim. Biophys. Acta 815, 468, 1985; or Sonnhammer et al., InJ. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C.Sensen, editors, Proceedings of the Sixth International Conference onIntelligent Systems for Molecular Biology, pages 175-182, Menlo Park,Calif., 1998. AAAI Press.).

Methods for the search and identification of RLK827 homologues would bewell within the realm of persons skilled in the art. Such methodscomprise comparison of the sequences represented by SEQ ID NO 1 or 2, ina computer readable format, with sequences that are available in publicdatabases such as MIPS (mips.gsf.de/webpage), GenBank(ncbi.nlm.nih.gov/Genbank/index.html webpage) or EMBL NucleotideSequence Database (ebi.ac.uk/embl/index.html webpage), using algorithmswell known in the art for the alignment or comparison of sequences, suchas GAP (Needleman and Wunsch, J. Mol. Biol. 48; 443-453 (1970)), BESTFIT(using the local homology algorithm of Smith and Waterman (Advances inApplied Mathematics 2; 482-489 (1981))), BLAST (Altschul, S. F., Gish,W., Miller, W., Myers, E. W. & Lipman, D. J., J. Mol. Biol. 215:403-410(1990)), FASTA and TFASTA (W. R. Pearson and D. J. LipmanProc.Natl.Acad.Sci. USA 85:2444-2448 (1988)). The software forperforming BLAST analysis is publicly available through the NationalCentre for Biotechnology Information (NCBI). The homologues mentionedbelow were identified using BLAST default parameters (BLOSUM62 matrix,gap opening penalty 11 and gap extension penalty 1) and preferably thefull-length sequences are used for analysis.

Examples of proteins falling under the definition of “RLK827 polypeptideor a homologue thereof” include the Arabidopsis proteins At1g51850,At1g51805, At1g51810, At2g04300, At3g21340, At1g49100. It should benoted that a cluster of related putative receptor like kinases arelocated in tandem on chromosome 1 of Arabidopsis thaliana, includingAt1g51800, At1g51805, At1g51810, At1g51820, At1g51830, At1g51840,At1g51850, At1g51860, At1g51870, At1g51880 and At1g51890, of which atleast four of them are highly related to RLK827.

It is to be understood that the term RLK827 polypeptide or a homologuethereof is not to be limited to the sequences represented by SEQ ID NO:2, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ IDNO: 17 and SEQ ID NO: 19, but that any polypeptide meeting the criteriaof (i) having a cytoplasmic kinase domain and (ii) having at least onebut no more than three LRR domains and preferably the consensus sequenceof SEQ ID NO: 33 in the putative non-cytoplasmic part of the protein,separated from the kinase domain by a transmembrane region, and whichkinase domain comprises the STYKc consensus sequence and/or (iii) beinga paralogue or orthologue of RLK827 and having at least 25% sequenceidentity to the sequence of SEQ ID NO: 2, may be suitable for use in themethods of the invention. Preferably, the kinase domain is functional,meaning that the RLK827 polypeptide or its homologue has kinaseactivity.

To determine the kinase activity of RLK827, several assays are availableand well known in the art (for example Current Protocols in MolecularBiology, Volumes 1 and 2, Ausubel et al. (1994), Current Protocols; oronline such as at the protocol-online org webpage). In brief, the kinaseassay generally involves (1) bringing the kinase protein into contactwith a substrate polypeptide containing the target site to bephosphorylated; (2) allowing phosphorylation of the target site in anappropriate kinase buffer under appropriate conditions; (3) separatingphosphorylated products from non-phosphorylated substrate after asuitable reaction period. The presence or absence of kinase activity isdetermined by the presence or absence of a phosphorylated target. Inaddition, quantitative measurements can be performed. Purified RLK827protein, or cell extracts containing or enriched in the RLK827 proteincould be used as source for the kinase protein. Alternatively, theapproach of Zhao et al. (Plant Mol. Biol. 26, 791-603, 1994) could beused, where the cytoplasmic domain of a rice receptor like kinase wasexpressed in Escherichia coli and assayed for kinase activity. As asubstrate, small peptides are particularly well suited. The peptide mustcomprise one or more serine, threonine or tyrosine residues in aphosphorylation site motif. A compilation of phosphorylation sites canbe found in Biochimica et Biophysica Acta 1314, 191-225, (1996). Inaddition, the peptide substrates may advantageously have a net positivecharge to facilitate binding to phosphocellulose filters, (allowing toseparate the phosphorylated from non-phosphorylated peptides and todetect the phosphorylated peptides). If a phosphorylation site motif isnot known, a general tyrosine kinase substrate can be used. For example,“Src-related peptide” (RRLIEDAEYAARG) is a substrate for many receptorand non-receptor tyrosine kinases). To determine the kinetic parametersfor phosphorylation of the synthetic peptide, a range of peptideconcentrations is required. For initial reactions, a peptideconcentration of 0.7-1.5 mM could be used. For each kinase enzyme, it isimportant to determine the optimal buffer, ionic strength, and pH foractivity. A standard 5× Kinase Buffer generally contains 5 mg/ml BSA(Bovine Serum Albumin preventing kinase adsorption to the assay tube),150 mM Tris-Cl (pH 7.5), 100 mM MgCl₂. Divalent cations are required formost tyrosine kinases, although some tyrosine kinases (for example,insulin-, IGF-1-, and PDGF receptor kinases) require MnCl₂ instead ofMgCl₂ (or in addition to MgCl₂). The optimal concentrations of divalentcations must be determined empirically for each protein kinase. Acommonly used donor for the phosphoryl group is radio-labelled[gamma-³²P]ATP (normally at 0.2 mM final concentration). The amount of³²P incorporated in the peptides may be determined by measuring activityon the nitrocellulose dry pads in a scintillation counter.

Alternatively, the activity of an RLK827 polypeptide or of a homologuethereof may be assayed by expressing the RLK827 polypeptide or of ahomologue thereof under control of a rice GOS2 promoter in rice plants,and in particular in the rice variety Nipponbare, which results inplants with increased yield compared to corresponding wild type plants.This increase in yield may for example be measured as one or more of anincrease in number of filled seeds, in total weight of seeds and/or inharvest index.

The nucleic acid encoding an RLK827 polypeptide or a homologue thereofmay be any natural or synthetic nucleic acid. An RLK827 polypeptide or ahomologue thereof as defined herein is encoded by an RLK827 nucleic acidmolecule. Therefore the term “RLK827 nucleic acid molecule” or “RLK827gene” as defined herein is any nucleic acid molecule encoding an RLK827polypeptide or a homologue thereof as defined above. Examples of RLK827nucleic acid molecules include those represented by any one of SEQ IDNO: 1, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16 and SEQ ID NO: 18, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQID NO 29, SEQ ID NO 31. RLK827 nucleic acids and functional variantsthereof may be suitable in practising the methods of the invention.Functional variant RLK827 nucleic acids include portions of an RLK827nucleic acid molecule and/or nucleic acids capable of hybridising withan RLK827 nucleic acid molecule or with a nucleic acid molecule encodinga homologue of RLK827. The term “functional” in the context of afunctional variant refers to a variant RLK827 nucleic acid molecule(i.e. a portion or a hybridising sequence), which encodes a polypeptidehaving kinase activity and comprising a non-cytoplasmic (extracellular)domain, which non-cytoplasmic domain comprises at least 1 but no morethan 3 LRR motifs and preferably also the amino acid sequence motif ofSEQ ID NO: 33 as defined above, and a C-terminal kinase domain, that isseparated from the non-cytoplasmic domain by a transmembrane domain.

The LRR-I type of receptor like kinases in plants have a modularstructure, and it has been shown that one LRR protein is able to binddifferent ligands, for example the tomato SR160 receptor and its tomatohomologue tBRH are able to bind brassinolide hormones and systemin, along distance signalling peptide. Brassinolide and systemin do notcompete for binding, suggesting they bind to different sites. Therefore,it is envisaged that engineering of LRR domains (e.g. by altering thenumber of LRR domains, or by performing domain stacking (binding to sameor different ligand(s)), or domain shuffling), in such a way that theactivity of the LRR is retained or modified, is useful in generatingvariant RLK827 nucleic acid molecules for performing the methods of theinvention. In a similar way, the kinase domain may be engineered toimprove kinase activity. A preferred type of variant includes thosegenerated by domain deletion, stacking or shuffling (see for example Heet al., Science 288, 2360-2363, 2000, or U.S. Pat. Nos. 5,811,238 and6,395,547).

The term portion as defined herein refers to a piece of DNA comprisingat least 150 nucleotides. A portion may be prepared, for example, bymaking one or more deletions to an RLK827 nucleic acid. The portions maybe used in isolated form or they may be fused to other coding (or noncoding) sequences in order to, for example, produce a protein thatcombines several activities, one of them being protein kinase activity.When fused to other coding sequences, the resulting polypeptide producedupon translation could be bigger than that predicted for the RLK827portion. The portion useful in the methods of the present inventioncomprises at least the kinase domain, preferably also a non-cytoplasmicLRR domain and a transmembrane domain located N-terminally of the kinasedomain, more preferably the portion comprises in the non-cytoplasmicdomain at least 1 but no more than 3 LRR domains, most preferably, theportion comprises in the non-cytoplasmic domain at least 1 but no morethan 3 LRR domains and the amino acid sequence motif of SEQ ID NO: 33 asdefined above. Preferably, the functional portion is a portion of anucleic acid as represented by any one of SEQ ID NO: 1, SEQ ID NO: 6,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ IDNO: 18.

The term “hybridisation” as defined herein is a process whereinsubstantially homologous complementary nucleotide sequences anneal toeach other. The hybridisation process can occur entirely in solution,i.e. both complementary nucleic acids are in solution. The hybridisationprocess can also occur with one of the complementary nucleic acidsimmobilised to a matrix such as magnetic beads, Sepharose beads or anyother resin. The hybridisation process can furthermore occur with one ofthe complementary nucleic acids immobilised to a solid support such as anitro-cellulose or nylon membrane or immobilised by e.g.photolithography to, for example, a siliceous glass support (the latterknown as nucleic acid arrays or microarrays or as nucleic acid chips).In order to allow hybridisation to occur, the nucleic acid molecules aregenerally thermally or chemically denatured to melt a double strand intotwo single strands and/or to remove hairpins or other secondarystructures from single stranded nucleic acids. The stringency ofhybridisation is influenced by conditions such as temperature, saltconcentration, ionic strength and hybridisation buffer composition.

“Stringent hybridisation conditions” and “stringent hybridisation washconditions” in the context of nucleic acid hybridisation experimentssuch as Southern and Northern hybridisations are sequence dependent andmay differ depending on environmental parameters. The skilled artisan isaware of various parameters which may be altered during hybridisationand washing and which will either maintain or change the stringencyconditions.

The T_(m) is the temperature under defined ionic strength and pH, atwhich 50% of the target sequence hybridises to a perfectly matchedprobe. The T_(m) is dependent upon the solution conditions and the basecomposition and length of the probe. For example, longer sequenceshybridise specifically at higher temperatures. The maximum rate ofhybridisation is obtained from about 16° C. up to 32° C. below T_(m).The presence of monovalent cations in the hybridisation solution reducethe electrostatic repulsion between the two nucleic acid strands therebypromoting hybrid formation; this effect is visible for sodiumconcentrations of up to 0.4M. Formamide reduces the melting temperatureof DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7° C. for each percentformamide, and addition of 50% formamide allows hybridisation to beperformed at 30 to 45° C., though the rate of hybridisation will belowered. Base pair mismatches reduce the hybridisation rate and thethermal stability of the duplexes. On average and for large probes, theT_(m), decreases about 1° C. per % base mismatch. The T_(m) may becalculated using the following equations, depending on the types ofhybrids:

DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984):

T _(m)=81.5° C.+16.6×log [Na⁺]^(a)+0.41×%[G/C ^(b)]−500×[L^(c)]⁻¹−0.61×% formamide

DNA-RNA or RNA-RNA hybrids:

T _(m)=79.8+18.5(log₁₀[Na⁺]^(a))+0.58 (% G/C ^(b))+11.8 (% G/C^(b))²−820/L^(c)

oligo-DNA or oligo-RNA^(d) hybrids:

For <20 nucleotides: T_(m)=2 (/_(n))

For 20-35 nucleotides: T_(m)=22+1.46 (/_(n))

^(a) or for other monovalent cation, but only accurate in the 0.01-0.4 Mrange.^(b) only accurate for % GC in the 30% to 75% range.^(c)L=length of duplex in base pairs.^(d) Oligo, oligonucleotide; /^(n), effective length of primer=2×(no. ofG/C)+(no. of A/T).

Note: for each 1% formamide, the T_(m) is reduced by about 0.6 to 0.7°C., while the presence of 6M urea reduces the T_(m) by about 30° C.

Specificity of hybridisation is typically the function ofpost-hybridisation washes. To remove background resulting fromnon-specific hybridisation, samples are washed with dilute saltsolutions. Critical factors of such washes include the ionic strengthand temperature of the final wash solution: the lower the saltconcentration and the higher the wash temperature, the higher thestringency of the wash. Wash conditions are typically performed at orbelow hybridisation stringency. Generally, suitable stringent conditionsfor nucleic acid hybridisation assays or gene amplification detectionprocedures are as set forth above. More or less stringent conditions mayalso be selected. Generally, low stringency conditions are selected tobe about 50° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and pH. Medium stringencyconditions are when the temperature is 20° C. below T_(m), and highstringency conditions are when the temperature is 10° C. below T_(m).For example, stringent conditions are those that are at least asstringent as, for example, conditions A-L; and reduced stringencyconditions are at least as stringent as, for example, conditions M-R.Non-specific binding may be controlled using any one of a number ofknown techniques such as, for example, blocking the membrane withprotein containing solutions, additions of heterologous RNA, DNA, andSDS to the hybridisation buffer, and treatment with Rnase.

Examples of hybridisation and wash conditions are listed in Table 1:

TABLE 1 Stringency Polynucleotide Hybrid Length HybridizationTemperature and Wash Temperature Condition Hybrid± (bp)‡ Buffer^(†) andBuffer^(†) A DNA:DNA > or 65° C. 1×SSC; or 42° C., 1×SSC 65° C.; 0.3×SSCequal to 50 and 50% formamide B DNA:DNA <50 Tb*; 1×SSC Tb*; 1×SSC CDNA:RNA > or 67° C. 1×SSC; or 45° C., 1×SSC 67° C.; 0.3×SSC equal to 50and 50% formamide D DNA:RNA <50 Td*; 1×SSC Td*; 1×SSC E RNA:RNA > or 70°C. 1×SSC; or 50° C., 1×SSC 70° C.; 0.3×SSC equal to 50 and 50% formamideF RNA:RNA <50 Tf*; 1×SSC Tf*; 1×SSC G DNA:DNA > or 65° C. 4×SSC; or 45°C.,.4×SSC 65° C.; 1×SSC equal to 50 and 50% formamide H DNA:DNA <150Th*; 4×SSC Th*; 4×SSC I DNA:RNA > or 67° C. 4×SSC; or 45° C., 4×SSC 67°C.; 1×SSC equal to 50 and 50% formamide J DNA:RNA <50 Tj*; 4×SSC Tj*;4×SSC K RNA:RNA > or 70° C. 4×SSC; or 40° C., 6×SSC 67° C.; 1×SSC equalto 50 and 50% formamide L RNA:RNA <50 TI*; 2×SSC TI*; 2×SSC M DNA:DNA >or 50° C. 4×SSC; or 40° C., 6×SSC 50° C.; 2×SSC equal to 50 and 50%formamide N DNA:DNA <50 Tn*; 6×SSC Tn*; 6×SSC 0 DNA:RNA > or 55° C.4×SSC; or 42° C., 6×SSC 55° C.; 2×SSC equal to 50 and 50% formamide PDNA:RNA <50 Tp*; 6×SSC Tp*; 6×SSC Q RNA:RNA > or 60° C. 4×SSC; or 45°C., 6×SSC 60° C.; 2×SSC equal to 50 and 50% formamide R RNA:RNA <50 Tr*;4×SSC Tr*; 4×SSC ‡The “hybrid length” is the anticipated length for thehybridising nucleic acid. When nucleic acids of known sequence arehybridised, the hybrid length may be determined by aligning thesequences and identifying the conserved regions described herein.^(†)SSPE (1×SSPE is 0.15M NaCl, 1O mM NaH₂PO₄, and 1.25 mM EDTA, pH7.4)may be substituted for SSC (1×SSC is 0.15M NaCI and 15 mM sodiumcitrate) in the hybridisation and wash buffers; washes are performed for15 minutes after hybridisation is complete. The hybridisations andwashes may additionally include 5 × Denhardt's reagent, 0.5-1.0% SDS,100 μg/ml denatured, fragmented salmon sperm DNA, 0.5% sodiumpyrophosphate, and up to 50% formamide. *Tb-Tr: The hybridisationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10° C. less than the melting temperature T_(m) of thehybrids; the T_(m) is determined according to the above-mentionedequations. ±The present invention also encompasses the substitution ofany one, or more DNA or RNA hybrid partners with either a PNA, or amodified nucleic acid.

For the purposes of defining the level of stringency, reference canconveniently be made to Sambrook et al. (2001) Molecular Cloning: alaboratory manual, 3^(rd) Edition Cold Spring Harbor Laboratory Press,CSH, New York or to Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989).

For example, a nucleic acid encoding SEQ ID NO: 2 or a homologue thereofmay be used in a hybridisation experiment. Alternatively fragmentsthereof may be used as probes. Depending on the starting pool ofsequences from which the RLK is to be identified, different fragmentsfor hybridization can be selected. For example, when a limited number ofhomologues with a high sequence identity to RLK827 are desired, a lessconserved fragment may be used for hybridisation such asGGTAGACTCGCCAAAGAATTTGAACCACTCGTTGAT (nucleotides 184 to 219 of SEQ IDNO: 1). By aligning SEQ ID NO 2 and homologues thereof it is possible todesign equivalent nucleic acid fragments useful as probes forhybridisation. Preferably the hybridising sequence comprises at leastthe kinase domain, preferably also a non-cytoplasmic LRR domain and atransmembrane domain located N-terminally of the kinase domain, morepreferably the portion comprises in the non-cytoplasmic domain at least1 but no more than 3 LRR domains, most preferably, the portion comprisesin the non-cytoplasmic domain at least 1 but no more than 3 LRR domainsand the amino acid sequence motif of SEQ ID NO: 33 as defined above.

After hybridisation and washing, the duplexes may be detected byautoradiography (when radiolabeled probes were used) or bychemiluminescence, immunodetection, by fluorescent or chromogenicdetection, depending on the type of probe labelling. Alternatively, aribonuclease protection assay may be performed for detection of RNA:RNAhybrids

The RLK827 nucleic acid molecule or variant thereof may be derived fromany natural or artificial source. The nucleic acid/gene or variantthereof may be isolated from a microbial source, such as bacteria, yeastor fungi, or from a plant, alga or animal (including human) source. Thisnucleic acid may be modified from its native form in composition and/orgenomic environment through deliberate human manipulation. The nucleicacid is preferably of plant origin, whether from the same plant species(for example to the one in which it is to be introduced) or whether froma different plant species. The nucleic acid may be isolated from adicotyledonous species, preferably from the family Brassicaceae, furtherpreferably from Arabidopsis thaliana. More preferably, the RLK827isolated from Arabidopsis thaliana is represented by SEQ ID NO: 1 andthe RLK827 amino acid sequence is as represented by SEQ ID NO: 2.

Functional variants useful in the methods of the present invention alsoinclude alternative splice variants of an RLK827 nucleic acid moleculeor gene. The term “alternative splice variant” as used hereinencompasses variants of a nucleic acid sequence in which selectedintrons and/or exons have been excised, replaced or added. Such variantswill be ones in which the biological activity of the protein isretained, which may be achieved by selectively retaining functionalsegments of the protein. Such splice variants may be found in nature ormay be manmade. Methods for making such splice variants are well knownin the art. Preferred splice variants are splice variants of a nucleicacid represented by SEQ ID NO: 1. Further preferred are splice variantsencoding a polypeptide retaining kinase activity and having at least onebut no more than three LRR domains in the putative non-cytoplasmic partof the protein, separated from the kinase domain by a transmembraneregion. More preferred splice variants comprise in addition also theamino acid sequence motif of SEQ ID NO: 33 in the putativenon-cytoplasmic domain. Most preferred splice variants of an RLK827nucleic acid molecule are those that encode an RLK827 polypeptide asdefined above.

Functional variants useful in the methods of the present inventionfurthermore include allelic variants of a nucleic acid encoding anRLK827 polypeptide or a homologue thereof, preferably an allelic variantof the nucleic acid represented by SEQ ID NO 1. Further preferably, thepolypeptide encoded by the allelic variant has kinase activity andretains at least one but no more than three LRR domains in the putativenon-cytoplasmic part of the protein, separated from the kinase domain bya transmembrane region. More preferred allelic variants comprise inaddition also the amino acid sequence motif of SEQ ID NO: 33 in theputative non-cytoplasmic domain. Most preferred allelic variants of anRLK827 nucleic acid molecule are those that encode an RLK827 polypeptideas defined above. Allelic variants exist in nature and encompassedwithin the methods of the present invention is the use of these naturalalleles. Allelic variants encompass Single Nucleotide Polymorphisms(SNPs), as well as Small Insertion/Deletion Polymorphisms (INDELs). Thesize of INDELs is usually less than 100 bp. SNPs and INDELs form thelargest set of sequence variants in naturally occurring polymorphicstrains of most organisms.

The expression and/or activity of an RLK827 polypeptide or a homologuethereof may also be increased by introducing a genetic modification(preferably in the locus of an RLK827 gene). The locus of a gene asdefined herein is taken to mean a genomic region which includes the geneof interest and 10 kb up- or downstream of the coding region.

The genetic modification may be introduced, for example, by any one (ormore) of the following methods: TDNA activation, TILLING, site-directedmutagenesis, homologous recombination, directed evolution or byintroducing and expressing in a plant a nucleic acid encoding an RLK827polypeptide or a homologue thereof. Following introduction of thegenetic modification there follows a step of selecting for increasedexpression and/or activity of an RLK827 polypeptide, which increase inexpression and/or activity gives plants having improved growthcharacteristics.

T-DNA activation tagging (Hayashi et al. Science 258, 1350-1353, 1992)involves insertion of T-DNA usually containing a promoter (may also be atranslation enhancer or an intron), in the genomic region of the gene ofinterest or 10 KB up- or down stream of the coding region of a gene in aconfiguration such that such promoter directs expression of the targetedgene. Typically, regulation of expression of the targeted gene by itsnatural promoter is disrupted and the gene falls under the control ofthe newly introduced promoter. The promoter is typically embedded in aT-DNA. This T-DNA is randomly inserted into a plant genome, for example,through Agrobacterium infection and leads to overexpression of genesnear to the inserted T-DNA. The resulting transgenic plants showdominant phenotypes due to overexpression of genes close to theintroduced promoter. The promoter to be introduced may be any promotercapable of directing expression of a gene in the desired organism, inthis case a plant. For example, constitutive, tissue-preferred, celltype-preferred and inducible promoters are all suitable for use in T-DNAactivation.

A genetic modification may also be introduced in the locus of an RLK827gene using the technique of TILLING (Targeted Induced Local Lesions INGenomes). This is a mutagenesis technology useful to generate and/oridentify, and to eventually isolate mutagenised variants of an RLK827nucleic acid molecule capable of exhibiting RLK827 activity. TILLINGalso allows selection of plants carrying such mutant variants. Thesemutant variants may even exhibit higher RLK827 activity than thatexhibited by the gene in its natural form. TILLING combines high-densitymutagenesis with high-throughput screening methods. The steps typicallyfollowed in TILLING are: (a) EMS mutagenesis (Redei and Koncz (1992),In: C Koncz, N-H Chua, J Schell, eds, Methods in Arabidopsis Research.World Scientific, Singapore, pp 16-82; Feldmann et al., (1994) In: E MMeyerowitz, C R Somerville, eds, Arabidopsis. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., pp 137-172; Lightner andCaspar (1998), In: J Martinez-Zapater, J Salinas, eds, Methods onMolecular Biology, Vol. 82. Humana Press, Totowa, N.J., pp 91-104); (b)DNA preparation and pooling of individuals; (c) PCR amplification of aregion of interest; (d) denaturation and annealing to allow formation ofheteroduplexes; (e) DHPLC, where the presence of a heteroduplex in apool is detected as an extra peak in the chromatogram; (f)identification of the mutant individual; and (g) sequencing of themutant PCR product. Methods for TILLING are well known in the art(McCallum Nature Biotechnol. 18, 455-457, 2000, Stemple Nature Rev.Genet. 5, 145-150, 2004).

Site-directed mutagenesis may be used to generated variants of RLK827nucleic acids or portions thereof that retain activity, namely, proteinkinase activity. Several methods are available to achieve site-directedmutagenesis, the most common being PCR based methods (See for exampleAusubel et al., Current Protocols in Molecular Biology. Wiley Eds., atthe 4ulr.com/products/currentprotocols/index.html webpage).

Directed evolution may be used to generate variants of RLK827 nucleicacid molecules or portions thereof encoding RKS11 or RKS4 polypeptidesor orthologues or portions thereof having an increased biologicalactivity. Directed evolution consists of iterations of DNA shufflingfollowed by appropriate screening and/or selection (Castle et al.,(2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and6,395,547).

TDNA activation, TILLING, site-directed mutagenesis and directedevolution are examples of technologies that enable the generation novelalleles and variants of RLK827 that retain RLK827 function and which aretherefore useful in the methods of the invention.

Homologous recombination allows introduction in a genome of a selectednucleic acid at a defined selected position. Homologous recombination isa standard technology used routinely in biological sciences for lowerorganism such as yeast or the moss Physcomitrella. Methods forperforming homologous recombination in plants have been described notonly for model plants (Offringa et al. (1990) EMBO J. 9, 3077-3084) butalso for crop plants, for example rice (Terada et al., (2002) NatureBiotechnol. 20, 1030-1034; or lida and Terada (2004) Curr. Opin.Biotechnol. 15, 132-138). The nucleic acid to be targeted (which may bean RLK827 nucleic acid molecule or variant thereof as hereinbeforedefined) need not be targeted to the locus of an RLK827 gene, but may beintroduced in, for example, regions of high expression. The nucleic acidto be targeted may be an improved allele used to replace the endogenousgene or may be introduced in addition to the endogenous gene.

According to a preferred embodiment of the invention, plant growthcharacteristics may be improved by introducing and expressing in a planta nucleic acid encoding an RLK827 polypeptide or a homologue thereof.

A preferred method for introducing a genetic modification (which in thiscase need not be in the locus of an RLK827 gene) is to introduce andexpress in a plant a nucleic acid encoding an RLK827 polypeptide or ahomologue thereof. An RLK827 polypeptide or homologue thereof asmentioned above is one having kinase activity and comprising anon-cytoplasmic (extracellular) domain, which non-cytoplasmic domaincomprises at least 1 but no more than 3 LRR motifs and preferably alsothe amino acid sequence motif of SEQ ID NO: 33 as defined above, and aC-terminal kinase domain that is separated from the non-cytoplasmicdomain by a transmembrane domain. Preferably, the RLK827 polypeptide orhomologue thereof has in increasing order of preference, at least 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%96%, 97%, 98% or 99% overall sequence identity to the amino acidsequence represented by SEQ ID NO: 2.

“Homologues” of a protein encompass peptides, oligopeptides,polypeptides, proteins and enzymes having amino acid substitutions,deletions and/or insertions relative to the unmodified protein inquestion and having similar biological and functional activity as theunmodified protein from which they are derived.

Encompassed by the term “homologues” are orthologous sequences andparalogous sequences, two special forms of homology which encompassevolutionary concepts used to describe ancestral relationships of genes.

The term “paralogous” relates to gene-duplications within the genome ofa species leading to paralogous genes. Paralogues of RLK827 may easilybe identified by performing a BLAST analysis against a set of sequencesfrom the same species as the query sequence.

The term “orthologous” relates to homologous genes in differentorganisms due to speciation. Orthologues in, for example, monocot plantspecies may easily be found by performing a so-called reciprocal blastsearch. This may be done by a first blast involving blasting thesequence in question (for example, SEQ ID NO: 1 or SEQ ID NO: 2) againstany sequence database, such as the publicly available NCBI databasewhich may be found at: the ncbi.nlm.nih.gov webpage. If orthologues inrice were sought, the sequence in question would be blasted against, forexample, the 28,469 full-length cDNA clones from Oryza sativa Nipponbareavailable at NCBI. BLASTn or tBLASTX may be used when starting fromnucleotides or BLASTP or TBLASTN when starting from the protein, withstandard default values. The blast results may be filtered. Thefull-length sequences of either the filtered results or the non-filteredresults are then blasted back (second blast) against the sequences ofthe organism from which the sequence in question is derived. The resultsof the first and second blasts are then compared. An orthologue is foundwhen the results of the second blast give as hits with the highestsimilarity an RLK827 nucleic acid or RLK827 polypeptide, for example, ifone of the organisms is Arabidopsis thaliana then a paralogue is found.In the case of large families, ClustalW may be used, followed by theconstruction of a neighbour joining tree, to help visualize theclustering. Using a reciprocal BLAST procedure a rice orthologue(Unigene accession number Os.26918) was identified represented by theESTs CB631540, CB628137.1 and CB31541.1. Preferred orthologues are thosehaving the highest similarity to RLK827 or to a paralogue thereof in areciprocal BLAST search. Other examples of rice orthologues are given inSEQ ID NOs 24, 26, 28, 30 and 32.

A homologue may be in the form of a “substitutional variant” of aprotein, i.e. where at least one residue in an amino acid sequence hasbeen removed and a different residue inserted in its place. Amino acidsubstitutions are typically of single residues, but may be clustereddepending upon functional constraints placed upon the polypeptide;insertions will usually be of the order of about 1 to 10 amino acidresidues. Preferably, amino acid substitutions comprise conservativeamino acid substitutions (Table 2). To produce such homologues, aminoacids of the protein may be replaced by other amino acids having similarproperties (such as similar hydrophobicity, hydrophilicity,antigenicity, propensity to form or break α-helical structures orβ-sheet structures). Conservative substitution tables are well known inthe art (see for example Creighton (1984) Proteins. W.H. Freeman andCompany).

TABLE 2 Examples of conserved amino acid substitutions: ResidueConservative Substitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Gln AsnCys Ser Glu Asp Gly Pro His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg;Gln Met Leu; Ile Phe Met; Leu; Tyr Ser Thr; Gly Thr Ser; Val Trp Tyr TyrTrp; Phe Val Ile; Leu

Less conserved substitutions can be made in case the above-mentionedamino acid properties are not so critical.

A homologue may also be in the form of an “insertional variant” of aprotein, i.e. where one or more amino acid residues are introduced intoa predetermined site in a protein. Insertions may compriseamino-terminal and/or carboxy-terminal fusions as well as intra-sequenceinsertions of single or multiple amino acids. Generally, insertionswithin the amino acid sequence will be smaller than amino- orcarboxy-terminal fusions, of the order of about 1 to 10 residues.Examples of amino- or carboxy-terminal fusion proteins or peptidesinclude the binding domain or activation domain of a transcriptionalactivator as used in the yeast two-hybrid system, phage coat proteins,(histidine)₆-tag, glutathione S-transferase-tag, protein A,maltose-binding protein, dihydrofolate reductase, Tag 100 epitope, c-mycepitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HAepitope, protein C epitope and VSV epitope.

Homologues in the form of “deletion variants” of a protein arecharacterised by the removal of one or more amino acids from a protein.

Amino acid variants of a protein may readily be made using peptidesynthetic techniques well known in the art, such as solid phase peptidesynthesis and the like, or by recombinant DNA manipulations. Methods forthe manipulation of DNA sequences to produce substitution, insertion ordeletion variants of a protein are well known in the art. For example,techniques for making mutations at predetermined sites in DNA are wellknown to those skilled in the art and include M 13 mutagenesis, T7-Genin vitro mutagenesis (USB, Cleveland, Ohio), QuickChange Site Directedmutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directedmutagenesis or other site-directed mutagenesis protocols.

The RLK827 polypeptide or homologue thereof may be a derivative.“Derivatives” include peptides, oligopeptides, polypeptides, proteinsand enzymes which may comprise substitutions, deletions or additions ofnaturally and non-naturally occurring amino acid residues compared tothe amino acid sequence of a naturally-occurring form of the protein,for example, as presented in SEQ ID NO 2. “Derivatives” of a proteinencompass peptides, oligopeptides, polypeptides, proteins and enzymeswhich may comprise naturally occurring altered, glycosylated, acylatedor non-naturally occurring amino acid residues compared to the aminoacid sequence of a naturally-occurring form of the polypeptide. Aderivative may also comprise one or more non-amino acid substituentscompared to the amino acid sequence from which it is derived, forexample a reporter molecule or other ligand, covalently ornon-covalently bound to the amino acid sequence, such as a reportermolecule which is bound to facilitate its detection, and non-naturallyoccurring amino acid residues relative to the amino acid sequence of anaturally-occurring protein.

According to a preferred aspect of the present invention, enhanced orincreased expression of an RLK827 nucleic acid molecule or variantthereof is envisaged. Methods for obtaining enhanced or increasedexpression of genes or gene products are well documented in the art andinclude, for example, overexpression driven by appropriate promoters,the use of transcription enhancers or translation enhancers. Isolatednucleic acids which serve as promoter or enhancer elements may beintroduced in an appropriate position (typically upstream) of anon-heterologous form of a polynucleotide so as to upregulate expressionof an RLK827 nucleic acid or variant thereof. For example, endogenouspromoters may be, altered in vivo by mutation, deletion, and/orsubstitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al.,PCT/US93/03868), or isolated promoters may be introduced into a plantcell in the proper orientation and distance from a gene of the presentinvention so as to control the expression of the gene.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region may be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added may be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

An intron sequence may also be added to the 5′ untranslated region orthe coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold (Buchman and Berg,Mol. Cell. Biol. 8, 4395-4405 (1988); Callis et al., Genes Dev. 1,1183-1200 (1987)). Such intron enhancement of gene expression istypically greatest when placed near the 5′ end of the transcriptionunit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1intron are known in the art. See generally, The Maize Handbook, Chapter116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

The invention also provides genetic constructs and vectors to facilitateintroduction and/or expression of the nucleotide sequences useful in themethods according to the invention.

Therefore, there is provided a gene construct comprising:

-   -   (i) an RLK827 nucleic acid molecule or functional variant        thereof;    -   (ii) one or more control sequence capable of driving expression        of the nucleic acid sequence of (i); and optionally    -   (iii) a transcription termination sequence.

Constructs useful in the methods according to the present invention maybe constructed using recombinant DNA technology well known to personsskilled in the art. The gene constructs may be inserted into vectors,which may be commercially available, suitable for transforming intoplants and suitable for expression of the gene of interest in thetransformed cells.

Plants are transformed with a vector comprising the sequence of interest(i.e., an RLK827 nucleic acid or functional variant thereof). Thesequence of interest is operably linked to one or more control sequences(at least to a promoter). The terms “regulatory element”, “controlsequence” and “promoter” are all used interchangeably herein and are tobe taken in a broad context to refer to regulatory nucleic acidsequences capable of effecting expression of the sequences to which theyare ligated. Encompassed by the aforementioned terms are transcriptionalregulatory sequences derived from a classical eukaryotic genomic gene(including the TATA box which is required for accurate transcriptioninitiation, with or without a CCAAT box sequence) and additionalregulatory elements (i.e. upstream activating sequences, enhancers andsilencers) which alter gene expression in response to developmentaland/or external stimuli, or in a tissue-specific manner. Also includedwithin the term is a transcriptional regulatory sequence of a classicalprokaryotic gene, in which case it may include a −35 box sequence and/or−10 box transcriptional regulatory sequences. The term “regulatoryelement” also encompasses a synthetic fusion molecule or derivativewhich confers, activates or enhances expression of a nucleic acidmolecule in a cell, tissue or organ. The term “operably linked” as usedherein refers to a functional linkage between the promoter sequence andthe gene of interest, such that the promoter sequence is able toinitiate transcription of the gene of interest.

Advantageously, any type of promoter may be used to drive expression ofthe nucleic acid sequence. The promoter may be an inducible promoter,i.e. having induced or increased transcription initiation in response toa developmental, chemical, environmental or physical stimulus. Anexample of an inducible promoter being a stress-inducible promoter, i.e.a promoter activated when a plant is exposed to various stressconditions, is the water stress induced promoter WS118. Additionally oralternatively, the promoter may be a tissue-specific promoter, i.e. onethat is capable of preferentially initiating transcription in certaintissues, such as the leaves, roots, seed tissue etc. An example of aseed-specific promoter is the rice oleosin 18 kDa promoter (Wu et al.(1998) J Biochem 123(3): 386-91).

Preferably, the RLK827 nucleic acid or functional variant thereof isoperably linked to a constitutive promoter. A constitutive promoter istranscriptionally active during most, but not necessarily all, phases ofits growth and development and is substantially ubiquitously expressed.Preferably, the constitutive promoter is a GOS2 promoter (from rice)(nucleotides 1 to 2193 in SEQ ID NO: 3). It should be clear that theapplicability of the present invention is not restricted to the RLK827nucleic acid represented by SEQ ID NO: 1, nor is the applicability ofthe invention restricted to expression of an RLK827 nucleic acid whendriven by a GOS2 promoter. Examples of other constitutive promoters thatmay also be used to drive expression of a RLK827 nucleic acid are shownin Table 3 below.

TABLE 3 Examples of constitutive promoters Gene Source Expression MotifReference Actin Constitutive McElroy et al, Plant Cell, 2: 163- 171,1990 CAMV 35S Constitutive Odell et al, Nature, 313: 810-812, 1985 CaMV19S Constitutive Nilsson et al., Physiol. Plant. 100: 456-462, 1997 GOS2Constitutive de Pater et al, Plant J Nov; 2(6): 837- 44, 1992 UbiquitinConstitutive Christensen et al, Plant Mol. Biol. 18: 675-689, 1992 Ricecyclophilin Constitutive Buchholz et al, Plant Mol Biol. 25(5): 837-43,1994 Maize H3 histone Constitutive Lepetit et al, Mol. Gen. Genet. 231:276-285, 1992 Actin 2 Constitutive An et al, Plant J. 10(1); 107-121,1996

Optionally, one or more terminator sequences may also be used in theconstruct introduced into a plant. The term “terminator” encompasses acontrol sequence which is a DNA sequence at the end of a transcriptionalunit which signals 3′ processing and polyadenylation of a primarytranscript and termination of transcription. Additional regulatoryelements may include transcriptional as well as translational enhancers.Those skilled in the art will be aware of terminator and enhancersequences which may be suitable for use in performing the invention.Such sequences would be known or may readily be obtained by a personskilled in the art.

An example of an expression cassette comprising the RLK827 nucleic acidoperably linked to the GOS2 promoter and further comprising a terminatorsequence is given in SEQ ID NO: 3.

The genetic constructs of the invention may further include an origin ofreplication sequence, which is required for maintenance and/orreplication in a specific cell type. One example is when a geneticconstruct is required to be maintained in a bacterial cell as anepisomal genetic element (e.g. plasmid or cosmid molecule). Preferredorigins of replication include, but are not limited to, the fl-ori andcolE1.

The genetic construct may optionally comprise a selectable marker gene.As used herein, the term “selectable marker gene” includes any genewhich confers a phenotype on a cell in which it is expressed tofacilitate the identification and/or selection of cells which aretransfected or transformed with a nucleic acid construct of theinvention. Suitable markers may be selected from markers that conferantibiotic or herbicide resistance, that introduce a new metabolic traitor that allow visual selection. Examples of selectable marker genesinclude genes conferring resistance to antibiotics (such as nptII thatphosphorylates neomycin and kanamycin, or hpt, phosphorylatinghygromycin), to herbicides (for example bar which provides resistance toBasta; aroA or gox providing resistance against glyphosate), or genesthat provide a metabolic trait (such as manA that allows plants to usemannose as sole carbon source). Visual marker genes result in theformation of colour (for example β-glucuranidase, GUS), luminescence(such as luciferase) or fluorescence (Green Fluorescent Protein, GFP,and derivatives thereof).

The present invention also encompasses plants obtainable by the methodsaccording to the present invention. The present invention thereforeprovides plants obtainable by the methods according to the presentinvention, which plants have introduced therein an RLK827 nucleic acidor a functional variant thereof, or which plants have introduced thereina genetic modification, preferably in the locus of an RLK827 gene.

The invention also provides a method for the production of transgenicplants having improved growth characteristics, comprising introductionand expression in a plant of an RLK827 nucleic acid or a functionalvariant thereof.

More specifically, the present invention provides a method for theproduction of transgenic plants having improved growth characteristics,which method comprises:

-   -   (i) introducing into a plant or plant cell an RLK827 nucleic        acid or a functional variant thereof; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.

The nucleic acid may: be introduced directly into a plant cell or intothe plant itself (including introduction into a tissue, organ or anyother part of a plant). According to a preferred feature of the presentinvention, the nucleic acid is preferably introduced into a plant bytransformation.

The term “transformation” as referred to herein encompasses the transferof an exogenous polynucleotide into a host cell, irrespective of themethod used for transfer. Plant tissue capable of subsequent clonalpropagation, whether by organogenesis or embryogenesis, may betransformed with a genetic construct of the present invention and awhole plant regenerated therefrom. The particular tissue chosen willvary depending on the clonal propagation systems available for, and bestsuited to, the particular species being transformed. Exemplary tissuetargets include leaf disks, pollen, embryos, cotyledons, hypocotyls,megagametophytes, callus tissue, existing meristematic tissue (e.g.,apical meristem, axillary buds, and root meristems), and inducedmeristem tissue (e.g., cotyledon meristem and hypocotyl meristem). Thepolynucleotide may be transiently or stably introduced into a host celland may be maintained non-integrated, for example, as a plasmid.Alternatively, it may be integrated into the host genome. The resultingtransformed plant cell may then be used to regenerate a transformedplant in a manner known to persons skilled in the art.

Transformation of plant species is now a fairly routine technique.Advantageously, any of several transformation methods may be used tointroduce the gene of interest into a suitable ancestor cell.Transformation methods include the use of liposomes, electroporation,chemicals that increase free DNA uptake, injection of the DNA directlyinto the plant, particle gun bombardment, transformation using virusesor pollen and microprojection. Methods may be selected from thecalcium/polyethylene glycol method for protoplasts (Krens et al. (1982)Nature 296, 72-74; Negrutiu et al. (1987) Plant Mol. Biol. 8, 363-373);electroporation of protoplasts (Shillito et al. (1985) Bio/Technol 3,1099-1102); microinjection into plant material (Crossway et al. (1986)Mol. Gen. Genet. 202, 179-185); DNA or RNA-coated particle bombardment(Klein et al. (1987) Nature 327, 70) infection with (non-integrative)viruses and the like. Transgenic rice plants expressing an RLK827 geneare preferably produced via Agrobacterium-mediated transformation usingany of the well known methods for rice transformation, such as describedin any of the following: published European patent application EP1198985 A1, Aldemita and Hodges (Planta 199, 612-617, 1996); Chan et al.(Plant Mol. Biol. 22, 491-506, 1993), Hiei et al. (Plant J. 6, 271-282,1994), which disclosures are incorporated by reference herein as iffully set forth. In the case of corn transformation, the preferredmethod is as described in either lshida et al. (Nature Biotechnol. 14,745-50, 1996) or Frame et al. (Plant Physiol. 129, 13-22, 2002), whichdisclosures are incorporated by reference herein as if fully set forth.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant.

Following DNA transfer and regeneration, putatively transformed plantsmay be evaluated, for instance using Southern analysis, for the presenceof the gene of interest, copy number and/or genomic organisation.Alternatively or additionally, expression levels of the newly introducedDNA may be monitored using Northern and/or Western analysis, bothtechniques being well known to persons having ordinary skill in the art.The cultivation of transformed plant cells into mature plants may thusencompass steps of selection and/or regeneration and/or growing tomaturity.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedto give homozygous second generation (or T2) transformants, and the T2plants further propagated through classical breeding techniques.

The generated transformed organisms may take a variety of forms. Forexample, they may be chimeras of transformed cells and non-transformedcells; clonal transformants (e.g., all cells transformed to contain theexpression cassette); grafts of transformed and untransformed tissues(e.g., in plants, a transformed rootstock grafted to an untransformedscion).

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant partsand propagules thereof. The present invention extends further toencompass the progeny of a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by any of theaforementioned methods, the only requirement being that progeny exhibitthe same genotypic and/or phenotypic characteristic(s) as those producedin the parent by the methods according to the invention. The inventionalso includes host cells containing an isolated RLK827 nucleic acid or afunctional variant thereof. Preferred host cells according to theinvention are plant cells. The invention also extends to harvestableparts of a plant according to the invention such as but not limited toseeds, leaves, fruits, flowers, stems, rhizomes, tubers and bulbs. Theinvention furthermore relates to products directly derived from aharvestable part of such a plant, such as dry pellets or powders, oil,fat and fatty acids, starch or proteins.

The present invention also encompasses the use of RLK827 nucleic acidsor functional variants thereof and to the use of RLK827 polypeptides orhomologues thereof.

One such use relates to improving the growth characteristics of plants.A preferred use relates to improving yield of plants, a more preferreduse relates to increasing seed yield. The seed yield may include one ormore of the following: increased number of (filled) seeds, increasedseed weight, increased harvest index, among others.

RLK827 nucleic acids or functional variants thereof or RLK827polypeptides or homologues thereof may find use in breeding programmesin which a DNA marker is identified which may be genetically linked toan RLK827 gene or variant thereof. The RLK827 or variants thereof orRLK827 or homologues thereof may be used to define a molecular marker.This DNA or protein marker may then be used in breeding programs toselect plants having altered growth characteristics. The RLK827 gene orvariant thereof may, for example, be a nucleic acid as represented byany one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23,SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 31.

Allelic variants of an RLK827 may also find use in marker-assistedbreeding programmes. Such breeding programmes sometimes requireintroduction of allelic variation by mutagenic treatment of the plants,using for example EMS mutagenesis; alternatively, the programme maystart with a collection of allelic variants of so called “natural”origin caused unintentionally. Identification of allelic variants thentakes place by, for example, PCR. This is followed by a selection stepfor selection of superior allelic variants of the sequence in questionand which give rise improved growth characteristics in a plant.Selection is typically carried out by monitoring growth performance ofplants containing different allelic variants of the sequence inquestion, for example, different allelic variants of any one of SEQ IDNO: 1, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ IDNO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 31. Growth performance may bemonitored in a greenhouse or in the field. Further optional stepsinclude crossing plants, in which the superior allelic variant wasidentified, with another plant. This could be used, for example, to makea combination of interesting phenotypic features.

An RLK827 nucleic acid or variant thereof may also be used as a probefor genetically and physically mapping the genes that they are a partof, and as markers for traits linked to those genes. Such informationmay be useful in plant breeding in order to develop lines with desiredphenotypes. Such use of RLK827 nucleic acids or variants thereofrequires only a nucleic acid sequence of at least 15 nucleotides inlength. The RLK827 nucleic acids or variants thereof may be used asrestriction fragment length polymorphism (RFLP) markers. Southern blotsof restriction-digested plant genomic DNA may be probed with the RLK827nucleic acids or variants thereof. The resulting banding patterns maythen be subjected to genetic analyses using computer programs such asMapMaker (Lander et al. (1987) Genomics 1, 174-181) in order toconstruct a genetic map. In addition, the nucleic acids may be used toprobe Southern blots containing restriction endonuclease-treated genomicDNAs of a set of individuals representing parent and progeny of adefined genetic cross. Segregation of the DNA polymorphisms is noted andused to calculate the position of the RLK827 nucleic acid or variantthereof in the genetic map previously obtained using this population(Botstein et al. (1980) Am. J. Hum. Genet. 32, 314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bematzky and Tanksley (Plant Mol. Biol. Reporter4, 37-41, 1986). Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

The nucleic acid probes may also be used for physical mapping (i.e., forthe placing of sequences on physical maps; see Hoheisel et al. In:Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996,pp. 319-346, and references cited therein).

In another embodiment, the nucleic acid probes may be used in directfluorescence in situ hybridization (FISH) mapping (Trask (1991) TrendsGenet. 7, 149-154). Although current methods of FISH mapping favour useof large clones (several to several hundred KB; see Laan et al. (1995)Genome Res. 5, 13-20), improvements in sensitivity may allow performanceof FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be earned out using the nucleic acids. Examplesinclude allele-specific amplification (Kazazian (1989) J. Lab. Clin.Med. 11, 95-96), polymorphism of PCR-amplified fragments (CAPS;Sheffield et al. (1993) Genomics 16, 325-332), allele-specific ligation(Landegren et al. (1988) Science 241, 1077-1080), nucleotide extensionreactions (Sokolov (1990) Nucleic Acid Res. 18, 3671), Radiation HybridMapping (Walter et al. (1997) Nat. Genet. 7, 22-28) and Happy Mapping(Dear and Cook (1989) Nucleic Acid Res. 17, 6795-6807). For thesemethods, the sequence of a nucleic acid is used to design and produceprimer pairs for use in the amplification reaction or in primerextension reactions. The design of such primers is well known to thoseskilled in the art. In methods employing PCR-based genetic mapping, itmay be necessary to identify DNA sequence differences between theparents of the mapping cross in the region corresponding to the instantnucleic acid sequence. This, however, is generally not necessary formapping methods.

In this way, generation, identification and/or isolation of modifiedplants with altered RLK827 expression and/or activity displayingimproved growth characteristics can be performed.

RLK827 nucleic acids or functional variants thereof or RLK827polypeptides or homologues thereof may also find use as growthregulators. Since these molecules have been shown to be useful inimproving the growth characteristics of plants, they would also beuseful growth regulators, such as herbicides or growth stimulators. Thepresent invention therefore provides a composition comprising an RLK827or a functional variant thereof or an RLK827 polypeptide or homologuethereof, together with a suitable carrier, diluent or excipient, for useas a growth regulator.

The methods according to the present invention result in plants havingimproved growth characteristics, as described hereinbefore. Theseadvantageous growth characteristics may also be combined with othereconomically advantageous traits, such as further yield-enhancingtraits, tolerance to various stresses, traits modifying variousarchitectural features and/or biochemical and/or physiological features.

DESCRIPTION OF FIGURES

The present invention will now be described with reference to thefollowing figures in which:

FIG. 1 gives a graphical overview of plant receptor like kinasestructures (adapted from Shiu & Bleecker, 2001). The arrow indicates thestructure of RLK827 and the subfamily to which RLK827 belongs.

FIG. 2 shows a schematic representation of the structure of SEQ ID NO:2. The triangle indicates sequence with an ATP-binding site signature.

FIG. 3 shows the binary vector p031 for transformation and expression inOryza sativa of an Arabidopsis thalianan RLK827 (internal referenceCDS0827) under the control of a rice GOS2 promoter (internal referencePR00129).

FIG. 4 A-V details examples of sequences useful in performing themethods according to the present invention. The “At” number refers tothe MIPs Accession number (mips.gsf.de web page); other identifiersrefer to GenBank accession numbers.

EXAMPLES

The present invention will now be described with reference to thefollowing examples, which are by way of illustration alone.

DNA manipulation: unless otherwise stated, recombinant DNA techniquesare performed according to standard protocols described in (Sambrook(2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold SpringHarbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubelet al. (1994), Current Protocols in Molecular Biology, Current Protocols(at the 4ulr.com/products/currentprotocols/index.html webpage). Standardmaterials and methods for plant molecular work are described in PlantMolecular Biology Labfax (1993) by R. D. D. Cray, published by BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications(UK).

Example 1 Gene Cloning

The Arabidopsis AtRLK827 (internal code CDS0827) was amplified by PCRusing as template an Arabidopsis thaliana seedling cDNA library(Invitrogen, Paisley, UK). After reverse transcription of RNA extractedfrom seedlings, the cDNAs were cloned into pCMV Sport 6.0. Averageinsert size of the bank was 1.5 kb, and the original number of cloneswas 1.59×10⁷ cfu. Original titer was determined to be 9.6×10⁵ cfu/ml,and after a first amplification of 6×10¹¹ cfu/ml. After plasmidextraction, 200 ng of template was used in a 50 μl PCR mix. Primersprm00405 (SEQ ID NO: 4, sense) and prm00406 (SEQ ID NO: 5, reversecomplementary), which include the AttB sites for Gateway recombination,were used for PCR amplification. PCR was performed using Hifi Taq DNApolymerase in standard conditions. A PCR fragment of 2750 bp wasamplified and purified also using standard methods. The first step ofthe Gateway procedure, the BP reaction, was then performed, during whichthe PCR fragment recombines in vivo with the pDONR201 plasmid toproduce, according to the Gateway® terminology, an “entry clone”, p3080.Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

Example 2 Vector Construction and Rice Transformation

The entry clone p3080 was subsequently used in an LR reaction with adestination vector used for Oryza sativa transformation. This vectorcontained as functional elements within the T-DNA borders: a plantselectable marker; a visual marker expression cassette; and a Gatewaycassette intended for LR in vivo recombination with the sequence ofinterest already cloned in the entry clone. A rice GOS2 promoter forconstitutive expression was located upstream of this Gateway cassette.

After the LR recombination step, the resulting expression vector p031(FIG. 3) was transformed into the Agrobacterium strain LBA4404 andsubsequently to Oryza sativa plants. Transformed rice plants wereallowed to grow and were then examined for the parameters described inExample 3.

Example 3 Evaluation of Trans Formants: Growth Measurements

Approximately 15 to 20 independent TO transformants were generated. Theprimary transformants were transferred from tissue culture chambers to agreenhouse for growing and harvest of T1 seed. Five events of which theT1 progeny segregated 3:1 for presence/absence of the transgene wereretained. For each of these events, 10 T1 seedlings containing thetransgene (hetero- and homo-zygotes), and 10 T1 seedlings lacking thetransgene (nullizygotes), were selected by visual marker screening. Theselected T1 plants were transferred to a greenhouse. Each plant receiveda unique barcode label to link unambiguously the phenotyping data to thecorresponding plant. The selected T1 plants were grown on soil in 10 cmdiameter pots under the following environmental settings:photoperiod=11.5 h, daylight intensity=30,000 lux or more, daytimetemperature=28° C. or higher, night time temperature=22° C., relativehumidity=60-70%. Transgenic plants and the corresponding nullizygoteswere grown side-by-side at random positions. From the stage of sowinguntil the stage of maturity the plants were passed several times througha digital imaging cabinet. At each time point digital images (2048×1536pixels, 16 million colours) were taken of each plant from at least 6different angles.

The mature primary panicles were harvested, bagged, barcode-labelled andthen dried for three days in the oven at 37° C. The panicles were thenthreshed and all the seeds collected. The filled husks were separatedfrom the empty ones using an air-blowing device. After separation, bothseed lots were then counted using a commercially available countingmachine. The empty husks were discarded. The filled husks were weighedon an analytical balance and the cross-sectional area of the seeds wasmeasured using digital imaging. This procedure resulted in the set ofseed-related parameters described below.

These parameters were derived in an automated way from the digitalimages using image analysis software and were analysed statistically. Atwo factor ANOVA (analyses of variance) corrected for the unbalanceddesign was used as statistical model for the overall evaluation of plantphenotypic characteristics. An F-test was carried out on all theparameters measured of all the plants of all the events transformed withthat gene. The F-test was carried out to check for an effect of the geneover all the transformation events and to verify for an overall effectof the gene, also named herein “global gene effect”. If the value of theF test shows that the data are significant, than it is concluded thatthere is a “gene” effect, meaning that not only presence or the positionof the gene is causing the effect. The threshold for significance for atrue global gene effect is set at 5% probability level for the F test.

To check for an effect of the genes within an event, i.e., for aline-specific effect, a t-test was performed within each event usingdata sets from the transgenic plants and the corresponding null plants.“Null plants” or “null segregants” or “nullizygotes” are the plantstreated in the same way as the transgenic plant, but from which thetransgene has segregated. Null plants can also be described as thehomozygous negative transformed plants. The threshold for significancefor the t-test is set at 10% probability level. The results for someevents can be above or below this threshold. This is based on thehypothesis that a gene might only have an effect in certain positions inthe genome, and that the occurrence of this position-dependent effect isnot uncommon. This kind of gene effect is also named herein a “lineeffect of the gene”. The p-value is obtained by comparing the t-value tothe t-distribution or alternatively, by comparing the F-value to theF-distribution. The p-value then gives the probability that the nullhypothesis (i.e., that there is no effect of the transgene) is correct.

The data obtained in the first experiment were confirmed in a secondexperiment with T2 plants. Three lines that had the correct expressionpattern were selected for further analysis. Seed batches from thepositive plants (both hetero- and homozygotes) in T1, were screened bymonitoring marker expression. For each chosen event, the heterozygoteseed batches were then retained for T2 evaluation. Within each seedbatch an equal number of positive and negative plants were grown in thegreenhouse for evaluation.

A total number of 120 RLK827 transformed plants were evaluated in the T2generation, that is 40 plants per event of which 20 positives for thetransgene, and 20 negatives.

Because two experiments with overlapping events have been carried out, acombined analysis was performed. This is useful to check consistency ofthe effects over the two experiments, and if this is the case, toaccumulate evidence from both experiments in order to increaseconfidence in the conclusion. The method used was a mixed-model approachthat takes into account the multilevel structure of the data (i.e.experiment—event—segregants). P-values are obtained by comparinglikelihood ratio test to chi square distributions.

Example 4 Evaluation of Transformants: Measurement of Seed-RelatedParameters

Upon analysis of the seeds as described above, the inventors found thatplants transformed with the RLK827 gene construct had a higher number offilled seeds, a higher total weight of seeds and an increased harvestindex compared to plants lacking the RLK827 transgene. Positive resultsobtained for plants in the T1 generation were again obtained in the T2generation. As an example, data for line OS2 are given (Table 4).

TABLE 4 Com- bined T1 generation T2 generation analysis Line OS2 %difference p-value % difference p-value p-value Nr filled seeds 410.0047 54 0.0726 0.0079 Total weight 43 0.0051 60 0.0655 0.0065 seedsHarvest Index 48 0.0007 57 0.0527 0.0003

Number of Filled Seeds:

The number of filled seeds was determined by counting the number offilled husks that remained after the separation step. Line OS2 showed asignificant increase in filled seed number of 41% for the T1 generation.This increase was also observed in the T2 generation (+54%). Thecombined analysis of T1 and T2 data confirmed that the effect on thenumber of filled seeds was highly significant (p-value of 0.0079).

Total Seed Yield:

The total seed yield (total weight of seeds) per plant was measured byweighing all filled husks harvested from a plant. Not only the number offilled seeds was increased, but also the total seed weight. In the firstgeneration there was an increase of 43%, which increase wasstatistically significant. This increase was confirmed in the T2generation and the combined analysis showed that the increases in seedyield were significant (p-value of 0.0065).

Harvest Index:

Line OS2 furthermore had an increased harvest index. The harvest indexin the present invention is defined as the ratio between the total seedyield and the above ground area (mm2), multiplied by a factor 106. Bothin T1 and T2 a positive effect on harvest index was observed (increaseof respectively 48 and 57% with p-values of 0.0007 and 0.0527). Heretoo, the combined analysis of the T1 and T2 data showed a significanteffect (p-value 0.0003).

1. A method for improving growth characteristics of a plant relative toa corresponding wild type plant, comprising increasing activity of anRLK827 polypeptide or a homologue thereof and/or by increasingexpression of an RLK827 encoding nucleic acid molecule, and optionallyselecting for plants having improved growth characteristics; wherein theRLK827 polypeptide or a homologue thereof comprises a non-cytoplasmicdomain having at least 1 but no more than 3 Leucine Rich Repeat (LRR)domains, a transmembrane domain, and a kinase domain.
 2. The method ofclaim 1, wherein said increased activity and/or increased expression iseffected by introducing a genetic modification in the locus of a geneencoding an RLK827 polypeptide or a homologue thereof.
 3. The method ofclaim 2, wherein said genetic modification is effected by one ofsite-directed mutagenesis, homologous recombination, TILLING, directedevolution and T-DNA activation. 4-10. (canceled)
 11. The method of claim1, wherein said improved plant growth characteristic is increased yield.12-13. (canceled)
 14. A plant or plant cell obtained by the method ofclaim
 1. 15. A construct comprising: (i) an RLK827 nucleic acid moleculeencoding a Receptor Like Kinase (RLK) comprising a non-cytoplasmicdomain having at least 1 but no more than 3 Leucine Rich Repeat (LRR)domains, a transmembrane domain, and a kinase domain; (ii) one or morecontrol sequence capable of driving expression of the nucleic acidsequence of (i); and optionally (iii) a transcription terminationsequence.
 16. The construct of claim 15, wherein said control sequenceis a constitutive promoter.
 17. The construct of claim 16, wherein saidconstitutive promoter is a GOS2 promoter.
 18. A plant or plant cellcomprising the construct of claim
 15. 19-25. (canceled)
 26. A method ofselecting a plant with improved growth characteristics comprisingutilizing an RLK827 nucleic acid molecule or functional variant thereofas a molecular marker.
 27. A composition comprising an RLK827 nucleicacid molecule or functional variant thereof or comprising an RLK827protein or a homologue thereof for improving growth characteristics ofplants, for use as a growth regulator.
 28. (canceled)
 29. The constructof claim 15, wherein said RLK827 nucleic acid molecule comprises asequence capable of hybridising under stringent conditions to thecomplementary strand of the RLK827 nucleic acid comprising the sequenceof SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ IDNO: 27, SEQ ID NO: 29, or SEQ ID NO: 31, wherein the stringentconditions comprise 1×SSC and 50% formamide at 65° C. or 42° C.,followed by washes at 65° C. in 0.3×SSC.
 30. The construct of claim 15,wherein the RLK827 nucleic acid molecule encodes an orthologue orparalogue of the RLK827 polypeptide which comprises a polypeptideencoded by the sequence of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO:
 31. 31.The construct of claim 15, wherein said RLK comprises a sequence havingat least 60% identity to the amino acid sequence of SEQ ID NO:
 2. 32.The construct of claim 15, wherein said RLK comprises a sequence havingat least 95% identity to the amino acid sequence of SEQ ID NO:
 2. 33.The construct of claim 15, wherein said RLK827 nucleic acid moleculeencodes a protein comprising a non-cytoplasmic domain with 1 but no morethan 3 LRR domains and the amino acid sequence motif of SEQ ID NO: 33, atransmembrane domain, and a kinase domain.
 34. The plant or plant cellof claim 18, wherein said plant is a monocotyledonous plant and whereinsaid plant cell is derived from a monocotyledonous plant.
 35. Aharvestable part, and/or a product directly derived therefrom, of theplant of claim 18, wherein said harvestable part and/or product comprisethe construct.
 36. The harvestable part of claim 35, wherein saidharvestable part is a seed.
 37. A method for increasing yield relativeto a corresponding wild type plant, comprising introducing andexpressing in a plant an RLK827 nucleic acid molecule encoding aReceptor Like Kinase (RLK) comprising a non-cytoplasmic domain having atleast 1 but no more than 3 Leucine Rich Repeat (LRR) domains, atransmembrane domain, and a kinase domain.
 38. The method of claim 37,wherein said RLK827 nucleic acid molecule comprises a sequence capableof hybridising under stringent conditions to the complementary strand ofthe RLK827 nucleic acid comprising the sequence of SEQ ID NO: 1, SEQ IDNO: 6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29,or SEQ ID NO: 31, wherein the stringent conditions comprise 1×SSC and50% formamide at 65° C. or 42° C., followed by washes at 65° C. in0.3×SSC.
 39. The method of claim 37, wherein said RLK827 nucleic acidmolecule is overexpressed in a plant.
 40. The method according to claim37, wherein said RLK827 nucleic acid molecule is of plant origin. 41.The method according to claim 37, wherein the RLK827 nucleic acidmolecule encodes an orthologue or paralogue of the RLK827 polypeptidewhich comprises a polypeptide encoded by the sequence of SEQ ID NO: 1,SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 29, or SEQ ID NO:
 31. 42. The method according to claim 37, whereinsaid RLK827 nucleic acid molecule is operably linked to a constitutivepromoter.
 43. The method according to claim 42, wherein saidconstitutive promoter is a GOS2 promoter.
 44. The method of claim 37,wherein said increased yield is increased seed yield.
 45. The methodaccording to claim 44, wherein said increased seed yield is selectedfrom any one or more of (i) increased seed biomass; (ii) increasednumber of (filled) seeds; (iii) increased seed size; (iv) increased seedvolume; (v) increased harvest index (HI); and (vi) increased thousandkernel weight (TKW).
 46. A plant or plant cell obtained by the methodaccording to claim
 37. 47. A method for the production of a transgenicplant having increased yield relative to a corresponding wild-typeplant, which method comprises: (i) introducing into a plant or plantcell an RLK827 nucleic acid molecule; and (ii) cultivating the plant orplant cell under conditions promoting plant growth and development. 48.A transgenic plant or plant cell having increased yield relative to acorresponding wild type plant resulting from an RLK827 nucleic acidmolecule introduced into said plant or plant cell, or resulting from agenetic modification in the locus of a gene encoding an RLK827polypeptide.
 49. The transgenic plant or plant cell according to claim46, wherein said plant is a monocotyledonous plant and wherein saidplant cell is derived from a monocotyledonous plant.
 50. A harvestablepart, and/or product directly derived therefrom, of a plant according toclaim 46, wherein said harvestable part and/or product comprise thenucleic acid molecule.
 51. The harvestable part of claim 50, whereinsaid harvestable part is a seed.
 52. The method of claim 37, whereinsaid RLK comprises a sequence having at least 44% identity to the aminoacid sequence of SEQ ID NO:
 2. 53. The method of claim 37, wherein saidRLK comprises a sequence having at least 60% identity to the amino acidsequence of SEQ ID NO:
 2. 54. The method of claim 37, wherein said RLKcomprises a sequence having at least 95% identity to the amino acidsequence of SEQ ID NO:
 2. 55. The method of claim 38, wherein saidRLK827 nucleic acid molecule encodes a protein comprising anon-cytoplasmic domain with 1 but no more than 3 LRR domains and theamino acid sequence motif of SEQ ID NO: 33, a transmembrane domain, anda kinase domain.
 56. A transgenic plant or plant cell having increasedyield relative to a corresponding wild type plant resulting from anRLK827 nucleic acid molecule introduced into said plant or plant cell.57. The transgenic plant or plant cell of claim 56, wherein said plantis a monocotyledonous plant and wherein said plant cell is derived froma monocotyledonous plant.
 58. A harvestable part, and/or a productdirectly derived therefrom, of the plant of claim 56, wherein saidharvestable part and/or product comprise the nucleic acid molecule. 59.The harvestable part of claim 58, wherein said harvestable part is aseed.
 60. The transgenic plant of claim 48, wherein the increased yieldis increased seed yield.
 61. The transgenic plant of claim 56, whereinthe increased yield is increased seed yield.